The overall goal of this project is to develop both potent and selective antifolates against Mycobacterium tuberculosis (Mtb), an opportunistic pathogen known to cause morbidity and mortality in AIDS patients. Specifically this project will focus on design of novel antifolates, through a structure-based approach in light of the recently solved crystal structure of Mtb dihydrofolate reductase (DHFR), organic synthesis, and evaluation of the synthetic compounds for their biological activity. DHFR is a key enzyme of the folate metabolic pathway and is required for both prokaryotic and eukaryotic cell-growth. Recent clinical studies of agents inhibiting enzymes of the folate pathway, including DHFR, demonstrated therapeutic effect in AIDS-associated TB patients. Highly potent DHFR inhibitors are available but are toxic due to their low selectivity. To increase the selectivity, a new pharmacophore model is needed, which relies critically on structural differences between the host and pathogen enzymes. The comparison between the available structures of the host and the pathogen DHFR enzymes has revealed potential target sites that allow for the design of selective inhibitors. With the common binding motifs of the DHFR family as platforms, such as 2,4-diaminopyrimidine and diaminopteridine heterocyclic systems, we have designed a series of molecules with special features that would bind tightly and specifically to these sites on Mtb DHFR but unlikely to the same sites on the human enzyme. In this proposed research, these designed molecules will be synthesized and evaluated for their inhibition of DHFR and their effect on Mtb cell growth. Lead compounds that actively and selectively inhibit Mtb will be co-crystallized with Mtb DHFR. The crystal structures of these complexes will be determined by X-ray crystallography. The structural analysis of these complexes will reveal the ligand binding mode to the target and the structural changes in the target induced by the binding of these compounds, which is critically useful for the lead-compound optimization. This study presents a new direction in the design of antifolates against mycobacterial infection. This study will also provide the molecular basis for further developing compounds that are highly potent and selective against Mtb and are potentially useful for the TB treatment.